The main goal of the research described in this proposal is to understand how long non-coding RNAs function in the cell. These RNAs are an important class of regulators of gene expression with incredibly diverse mechanisms of action. One of the most intriguing among these is their concerted action with the Polycomb Repressive Complex 2 (PRC2), a histone H3K27 methyltransferase complex, to regulate the expression of important developmental and proliferation factors. Despite their critical importance, how lncRNAs and PRC2 exert regulation of the epigenome in a cell- and developmentally specific manner is unknown. This proposal will address three complementary questions into the function of lncRNAs: How does PRC2 recognize its RNA targets? What is the role of internal adenosine methylation in the stability and function of lncRNAs? What is the role of RNA adenosine methylation in the execution of cellular developmental programs? The first aim will investigate the physical conformations adopted by lncRNAs that are bound by PRC2, and how this recognition may be affected by RNA modification. This will be carried out using a combination of tools to probe the structure of RNAs, RNA-protein binding, and cutting-edge next-generation sequencing technologies that will enable this examination in a transcriptome-wide scale. The second aim will examine the role of RNA adenosine methylation in the function and stability of two well-characterized lncRNAs, Xist and HOTAIR. RNA methylation activity in vivo will be downregulated and its consequences in the function and stability of these transcripts will be assessed. The final aim of this proposal will constitute the beginning of a long-term, independent investigation into the factors that regulate the function of lncRNAs in cell differentiation and the maintenance of the pluripotent state of stem cells. The work outlined here will shed light into the complex and poorly understood interplay of processes that control the timing of gene expression profiles, the development, establishment, and maintenance of cell identity, as well as serve as groundwork to understand when they go awry in disease. Statement of